Prostaglandins, a family of potent biological agents derived from the essential fatty acids, have recently been implicated as mediators of intercellular responses possibly via interaction with the nucleotide cyclase systems. In this project we propose to use previously developed tissue culture procedures to study in depth prostaglandin synthesis from labelled essential fatty acid precursors in isolated cells. Since the previous application was submitted, Samuelsson et al. have reported that transformation of the BHK cell line by oncogenic viruses results in marked changes in both the absolute level and the pattern of prostaglandins produced. As an immediate goal this study will concentrate on confirming these findings. Procedures for isolation and determination of small quantities of prostaglandins synthesized from essential fatty acid precursors of high specific activity have now been worked out. The method involves carrier dilution, ether extraction derivatization and isolation by gas liquid chromatography using an instrument equipped with sensitive dual mass and radioisotope monitors. It has been validated by determination of 0.1 nanogram quantities of H3-PGE2 and PGF2 alpha in tissue culture fluids with a precision of about plus or minus 5%. These procedures will be used to characterize prostaglandin production by a number of diploid cell lines and their transformed counterparts grown under controlled conditions. If the marked changes noted by Samuelsson are confirmed, then the investigation will focus on identifying the enzymatic defect in the prostaglandin regulating mechanism in transformed cells. It is expected that these studies will provide basic information on the factors which regulate prostaglandin synthesis at the cellular level. This is an area where very little is known at present, and where more information is badly needed.